Introduction
Synapse formation is a critical step in the assembly of neuronal circuits. Both secreted and membrane-associated proteins contribute to the assembly and maturation of synapses. In addition, vitamins, including folate and genes, have been suggested to play an important role in synaptogenesis. Folate is essential for the development and proper functioning of the nervous system. Getting enough folic acid (FA), the synthetic form of folate, at the time of conception lowers the risk for most neural tube defects in the offspring. It has been demonstrated that periconceptional FA supplementation promotes synaptic plasticity in male rat offspring and exerts long-term beneficial effects on cognitive performance. Moreover, some studies revealed that FA had a positive impact on the recovery of learning and memory abilities through alleviating synapse loss and synaptic dysfunction in Alzheimer’s disease. 
Many genes have been shown to be involved in the differentiation, proliferation, and migration of neurons during brain development. For example, during neuronal migration, growth, and axon guidance, there are cytoskeletal proteins known as navigators. The neuron navigator 1 (Nav1) is involved in neuronal migration and neuronal signaling pathways that guide axons in the appropriate direction. Other genes involved in synapse formation include the neuroligin family members, which may form trans-synaptic contacts with presynaptic neurexins. Neuroligins are essential for the proper maturation of synapses and brain function by recruiting scaffolding proteins, postsynaptic receptors, and signaling proteins. neuroligin1 (Nlgn1) is a cell adhesion molecule involved in the formation of synapses. The aim of this study was to evaluate the effect of normal and high doses of FA during pregnancy on the expression of Nav1 and Nlgn1 genes in the cerebral cortex of newborn mice.

Methods
Forty-five pregnant BALB/c mice were divided into three groups (15 per group). The first and second groups received FA at doses of 2 mg/kg/BW (normal) and 40 mg/kg/BW (high) daily during pregnancy, respectively. The third group did not receive FA and was considered the control group. Neonatal cerebral cortex was collected for the expression analysis of Nav1 and Nlgn1 genes using real-time PCR.

Results
Based on the results of the Nanodrop device, the concentration of total RNA in most samples was in the range of 600-900 ng/μl, and the absorbance ratio at 260/280 nm (which is an indicator for examining purity, lack of contamination, and quality of RNA) was in the range of 1.8-2. In order to examine the quality of the extracted RNAs, they were examined by agarose gel electrophoresis, and the rRNA bands of 18s, 28s, and 8/5s were observed, indicating good quality of the RNAs. The expression level of the Nav1 gene in the group treated with 2 mg/kg/BW FA was 1.48±0.08, which was significantly higher than in the control group (1.001±0.05) (P<0.001). The expression level of this gene in the group treated with 40 mg/kg/BW FA was 0.20±0.02, which was significantly lower than in the control group (P<0.001). Also, a significant difference in Nav1 gene expression was observed between the normal and high-dose FA groups (P<0.0001). Similar results were observed for the Nlgn1 gene. The expression of this gene in the normal-dose FA group was significantly higher than in the control group (1.21±0.02 vs 1.001±0.04, P<0.001). In the high-dose FA group, the expression level was significantly lower than in the control group (0.13±0.03, P<0.001). 
The protein-protein interaction was also examined by bioinformatics analysis. A network of 100 proteins related to Nlgn1 and Nav1 was mapped and functionally enriched to find the most relevant biological processes, molecular functions, cellular structures, and kyoto encyclopedia of genes and genomes (KEGG) pathways. The two genes were connected to each other through protein or functional interactions, as well as through synaptic regulation. 

Conclusion
The results of this study showed that the normal dose of DA can increase the expression of Nav1 and Nlgn1 genes in the cerebral cortex of newborn mice. However, the high dose of FA reduces the expression of both genes. One of the reasons for the decrease in gene expression after injection of high doses of FA is probably due to the increase in the level of gene promoter methylation by the methyl donor, folic acid. High doses of FA cause the methylation of gene promoters, a mechanism that suppresses gene expression. Additionally, the protein-protein interaction revealed the relationship between the two Nav1 and Nlgn1 proteins, highlighting the crucial role of these two proteins in synapses and ultimately in brain development.

Ethical Considerations

Compliance with ethical guidelines
This study was approved by the Ethics Committee of the University of Guilan, Rasht, Iran (Code: IR.GUILAN.REC.1403.027).

Funding
This study was funded by the University of Guilan, Rasht, Iran. 

Authors contributions
Conceptualization, study design, funding acquisition, critical revision and supervision: Farhad Mashayekhi; preparing the initial draft and statistical analysis: Zahra Khoshkar Chalaksarei; Data analysis and interpretation, administrative, technical, or material support: All authors.

Conflicts of interest
The authors declared no conflicts of interest.

Acknowledgments
The authors would like to thank the Vice-Chancellor for Research of the University of Guilan for providing financial support.



 

1. Stiles J, Jernigan TL. The basics of brain development. Neuropsychology Review. 2010; 20(4):327-48. [DOI:10.1007/s11065-010-9148-4] [PMID] [PMCID] 
2. Schepanski S, Buss C, Hanganu-Opatz IL, Arck PC. Prenatal immune and endocrine modulators of offspring's brain development and cognitive functions later in life. Frontiers in Immunology. 2018; 9:2186. [DOI:10.3389/fimmu.2018.02186] [PMID] [PMCID] 
3. Kwan KY, Sestan N, Anton ES. Transcriptional co-regulation of neuronal migration and laminar identity in the neocortex. Development. 2012; 139(9):1535-46. [DOI:10.1242/dev.069963] [PMID] [PMCID] 
4. Grützner N, Opriessnig T, Lopes R, Suchodolski JS, Nathues H, Steiner JM. Assessment of folate and cobalamin concentrations in relation to their dependent intracellular metabolites in serum of pigs between 6 and 26 weeks of age. Research in Veterinary Science. 2020; 130:59-67. [DOI:10.1016/j.rvsc.2020.02.002] [PMID] 
5. Imbard A, Benoist JF, Blom HJ. Neural tube defects, folic acid and methylation. International Journal of Environmental Research and Public Health. 2013; 10(9):4352-89. [DOI:10.3390/ijerph10094352] [PMID] [PMCID] 
6. Harlan De Crescenzo A, Panoutsopoulos AA, Tat L, Schaaf Z, Racherla S, et al. Deficient or excess folic acid supply during pregnancy alter cortical neurodevelopment in mouse offspring. Cerebral Cortex. 2021; 31(1):635-49. [DOI:10.1093/cercor/bhaa248] [PMID] [PMCID] 
7. Virdi S, Jadavji NM. The impact of maternal folates on brain development and function after birth. Metabolites. 2022; 12(9):876. [DOI:10.3390/metabo12090876] [PMID] [PMCID] 
8. Sandeep P, Sharma P, Luhach K, Dhiman N, Kharkwal H, Sharma B. Neuron navigators: A novel frontier with physiological and pathological implications. Molecular and Cellular Neurosciences. 2023; 127:103905. [DOI:10.1016/j.mcn.2023.103905] [PMID] 
9. Powers RM, Hevner RF, Halpain S. The neuron navigators: Structure, function, and evolutionary history. Frontiers in Molecular Neuroscience. 2023; 15:1099554. [DOI:10.3389/fnmol.2022.1099554] [PMID] [PMCID] 
10. Powers RM, Daza R, Koehler AE, Courchet J, Calabrese B, Hevner RF, et al. Growth cone macropinocytosis of neurotrophin receptor and neuritogenesis are regulated by neuron navigator 1. Molecular Biology of the Cell. 2022; 33(7):ar64. [DOI:10.1091/mbc.E21-12-0623] [PMID] [PMCID] 
11. van Haren J, Boudeau J, Schmidt S, Basu S, Liu Z, Lammers D, et al. Dynamic microtubules catalyze formation of navigator-TRIO complexes to regulate neurite extension. Current Biology. 2014; 24(15):1778-85. [DOI:10.1016/j.cub.2014.06.037] [PMID] 
12. Wang J, Sudhof T, Wernig M. Distinct mechanisms control the specific synaptic functions of neuroligin 1 and neuroligin 2. EMBO Reports. 2025; 26(3):860-79. [DOI:10.1038/s44319-024-00286-4] [PMID] [PMCID] 
13. Nguyen TA, Lehr AW, Roche KW. Neuroligins and neurodevelopmental disorders: X-linked genetics. Frontiers in Synaptic Neuroscience. 2020; 12:33. [DOI:10.3389/fnsyn.2020.00033] [PMID] [PMCID] 
14. Lisé MF, El-Husseini A. The neuroligin and neurexin families: From structure to function at the synapse. Cellular and Molecular Life Sciences. 2006; 63(16):1833-49. [DOI:10.1007/s00018-006-6061-3] [PMID] [PMCID] 
15. Trobiani L, Meringolo M, Diamanti T, Bourne Y, Marchot P, Martella G, et al. The neuroligins and the synaptic pathway in Autism Spectrum Disorder. Neuroscience and Biobehavioral Reviews. 2020; 119:37-51. [DOI:10.1016/j.neubiorev.2020.09.017] [PMID] 
16. Greenberg JA, Bell SJ, Guan Y, Yu YH. Folic Acid supplementation and pregnancy: More than just neural tube defect prevention. Reviews in Obstetrics and Gynecology. 2011; 4(2):52-9. [DOI:10.18370/2309-4117.2017.34.57-63] 
17. Giovedí S, Corradi A, Fassio A, Benfenati F. Involvement of synaptic genes in the pathogenesis of autism spectrum disorders: the case of synapsins. Frontiers in Pediatrics. 2014; 2:94. [DOI:10.3389/fped.2014.00094] [PMID] [PMCID] 
18. Jang S, Lee H, Kim E. Synaptic adhesion molecules and excitatory synaptic transmission. Current Opinion in Neurobiology. 2017; 45:45-50. [DOI:10.1016/j.conb.2017.03.005] [PMID] 
19. Huang Y, He Y, Sun X, He Y, Li Y, Sun C. Maternal high folic acid supplement promotes glucose intolerance and insulin resistance in male mouse offspring fed a high-fat diet. International Journal of Molecular Sciences. 2014; 15(4):6298-313. [DOI:10.3390/ijms15046298] [PMID] [PMCID] 
20. Naninck EFG, Stijger PC, Brouwer-Brolsma EM. The importance of maternal folate status for brain development and function of offspring. Advances in Nutrition. 2019; 10(3):502-19. [DOI:10.1093/advances/nmy120] [PMID] [PMCID] 
21. Sun WX, Shu YP, Yang XY, Huang W, Chen J, Yu NN, et al. Effects of folic acid supplementation in pregnant mice on glucose metabolism disorders in male offspring induced by lipopolysaccharide exposure during pregnancy. Scientific Reports. 2023; 13(1):7984. [DOI:10.1038/s41598-023-31690-w] [PMID] [PMCID] 
22. Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behavior Research Methods. 2007; 39(2):175-91. [DOI:10.3758/BF03193146] [PMID] 
23. van de Rest O, van Hooijdonk LW, Doets E, Schiepers OJ, Eilander A, de Groot LC. B vitamins and n-3 fatty acids for brain development and function: Review of human studies. Annals of Nutrition & Metabolism. 2012; 60(4):272-92. [DOI:10.1159/000337945] [PMID] 
24. Greene ND, Copp AJ. Neural tube defects. Annual Review of Neuroscience. 2014; 37:221-42. [DOI:10.1146/annurev-neuro-062012-170354] [PMID] [PMCID] 
25. Douet V, Chang L, Cloak C, Ernst T. Genetic influences on brain developmental trajectories on neuroimaging studies: From infancy to young adulthood. Brain Imaging and Behavior. 2014; 8(2):234-50. [DOI:10.1007/s11682-013-9260-1] [PMID] [PMCID] 
26. Wang D, Xu Y, Hong L, Qi B, Li X, Xie C, et al. Expose to high doses of folic acid during pregnancy causes adolescent anxiety- and depression-like behaviors in offspring mice. Journal of Affective Disorders. 2025; 368:770-8. [DOI:10.1016/j.jad.2024.09.105] [PMID] 
27. Heydari B, Mashayekhi F, Kashani MHG. Effect of in ovo feeding of folic acid on Disabled-1 and gga-miR-182-5p expression in the cerebral cortex of chick embryo. Journal of Animal Physiology and Animal Nutrition. 2024; 108(2):285-90. [DOI:10.1111/jpn.13889] [PMID] 
28. Liang X, Shi L, Wang M, Zhang L, Gong Z, Luo S, et al. Folic acid ameliorates synaptic impairment following cerebral ischemia/reperfusion injury via inhibiting excessive activation of NMDA receptors. The Journal of Nutritional Biochemistry. 2023; 112:109209. [DOI:10.1016/j.jnutbio.2022.109209] [PMID] 
29. Razeghi E, Mashayekhi F, Ghasemian F, Salehi Z. Effect of in ovo feeding of folic acid on brain derived neurotrophic factor (BDNF) and gga-miR-190a-3p expression in the developing cerebral cortex of chickens. Research in Veterinary Science. 2023; 154:73-77. [DOI:10.1016/j.rvsc.2022.11.010] [PMID] 
30. Luan Y, Cosín-Tomás M, Leclerc D, Malysheva OV, Caudill MA, Rozen R. Moderate folic acid supplementation in pregnant mice results in altered sex-specific gene expression in brain of young mice and embryos. Nutrients. 2022; 14(5):1051. [DOI:10.3390/nu14051051] [PMID] [PMCID] 
31. Li W, Li Z, Zhou D, Zhang X, Yan J, Huang G. Maternal folic acid deficiency stimulates neural cell apoptosis via miR-34a associated with Bcl-2 in the rat foetal brain. International Journal of Developmental Neuroscience. 2019; 72:6-12. [DOI:10.1016/j.ijdevneu.2018.11.002] [PMID] 
32. Henzel KS, Ryan DP, Schröder S, Weiergräber M, Ehninger D. High-dose maternal folic acid supplementation before conception impairs reversal learning in offspring mice. Scientific Reports. 2017; 7(1):3098. [DOI:10.1038/s41598-017-03158-1] [PMID] [PMCID] 
33. Partearroyo T, Pérez-Miguelsanz J, Peña-Melián Á, Maestro-de-Las-Casas C, Úbeda N, Varela-Moreiras G. Low and high dietary folic acid levels perturb postnatal cerebellar morphology in growing rats. The British Journal of Nutrition. 2016; 115(11):1967-77. [DOI:10.1017/S0007114516001008] [PMID] 
34. Barua S, Kuizon S, Chadman KK, Brown WT, Junaid MA. Microarray analysis reveals higher gestational folic Acid alters expression of genes in the cerebellum of mice offspring-a pilot study. Brain Sciences. 2015; 5(1):14-31. [DOI:10.3390/brainsci5010014] [PMID] [PMCID] 
35. Barua S, Chadman KK, Kuizon S, Buenaventura D, Stapley NW, Ruocco F, Begum U, et al. Increasing maternal or post-weaning folic acid alters gene expression and moderately changes behavior in the offspring. Plos One. 2014; 9(7):e101674. [DOI:10.1371/journal.pone.0101674] [PMID] [PMCID] 
36. Girotto F, Scott L, Avchalumov Y, Harris J, Iannattone S, Drummond-Main C, et al. High dose folic acid supplementation of rats alters synaptic transmission and seizure susceptibility in offspring. Scientific Reports. 2013; 3:1465. [DOI:10.1038/srep01465] [PMID] [PMCID] 
37. Mikael LG, Deng L, Paul L, Selhub J, Rozen R. Moderately high intake of folic acid has a negative impact on mouse embryonic development. Birth Defects Research. Part A, Clinical and Molecular Teratology. 2013; 97(1):47-52. [DOI:10.1002/bdra.23092] [PMID] 
38. Kucha W, Seifu D, Tirsit A, Yigeremu M, Abebe M, Hailu D, et al. Folate, vitamin B12, and homocysteine levels in women with neural tube defect-affected pregnancy in Addis Ababa, Ethiopia. Frontiers in Nutrition. 2022; 9:873900. [DOI:10.3389/fnut.2022.873900] [PMID] [PMCID] 
39. Kiselev IS, Kulakova OG, Boyko AN, Favorova OO. DNA methylation as an epigenetic mechanism in the development of multiple sclerosis. Acta Naturae. 2021; 13(2):45-57. [DOI:10.32607/actanaturae.11043] [PMID] [PMCID]